DESCRIPTION (Adapted from the abstract provided by the applicant): It is becoming increasingly apparent that mitochondria play a critical role in a wide range of acute and chronic neurodegenerative diseases. Recent studies from this laboratory and many others have suggested that mitochondria may be the source of a signal that kills neurons. Perhaps the best candidate for this signal is mitochondrial generation of reactive oxygen species (ROS). The mechanisms of ROS generation by brain mitochondria, however, are poorly understood. Moreover, little is known about the regulation of the ROS signal. We propose to investigate the characteristics of ROS generation by brain mitochondria and cultured neurons. In Specific Aim 1 we will determine the site(s) in the electron transport chain (ETC) that are the source of superoxide, and will determine the consequence of ETC inhibition on ROS generation in isolated brain mitochondria. Thus we will be able to model neurodegenerative disease based on prior reports of ETC inhibition in disorders such as Parkinson's disease and Alzheimer's disease. In Specific Aim 2 we will investigate the mechanisms that regulate ROS generation and will focus on agents that alter the mitochondrial membrane potential and agents that uncouple oxygen consumption from ATP synthesis. These agents include calcium and substrates for uncoupling proteins. In Specific Aim 3 we will use a series of neuron preparations to establish the properties of ROS generation by mitochondria in their intact cellular environment. We will use standard neuronal cultures, permeabilized neurons and acutely dissociated cells, along with fluorescence imaging of ROS, to establish neuron-specific mechanisms of mitochondrial ROS generation. In the final Specific Aim we will investigate whether the retrograde transport of mitochondria by neurons in vivo is altered by oxidative injury. This novel approach will allow us to investigate the properties of cellular mitochondrial homeostasis which will provide a long-term view of the role of mitochondria in neurodegeneration. These studies will illuminate a critical role of mitochondria and ROS signaling in neuronal injury, and will provide novel targets for intervention in a wide range of neurodegenerative disease.